Stably transfected Kc cells expressing a fluorescent insulator protein construct will be subjected to RNAi knockdown using a library of 21,000 dsRNAs corresponding to all annotated Drosophila genes. Automated imaging will be utilized to identify RNAs that no longer support GFP reporter localization to 20-25 nuclear foci indicating disruption of insulator body formation. This screen should identify factors required specifically for insulator organization as well as general nuclear organization. We will perform the screen with the assistance of the Drosophila RNAi Screening Center at Harvard Medical School.[unreadable] [unreadable] Genes required for RNAi are themselves susceptible to dsRNA knockdown. The precise functions of a number of factors required for RNA silencing, particularly RNA helicases, are not known, and there are some genes for which no mutants exist. These cell-based experiments will allow us to test rapidly each known component of the RNA silencing pathways for their effects on insulator function in order to better understand the overlap of these two pathways. Finally, this assay can be used to test novel factors that we have found previously to copurify with insulator proteins, such as an uncharacterized protein that harbors similarity to a nuclear pore protein with potential involvement in the tethering insulator bodies to the nuclear periphery.